RNA qPCR probes

Metabion's RNA dual labelled probes (RNA DLPs) are optimized for use in real-time quantitative PCR. Moreover, our RNA DLPs are produced according to technologically advanced synthesis and purification protocols, to ensure you that the eventual background noise will be reduced to an absolute minimum.

Our range of reporter-quencher combinations suit probe-hydrolysis as well as probe-hybridisation based assays. We believe this will cover your needs for single or multiplex real-time PCR applications.

We offer our RNA DLPs in final yields in nmoles. With this, we introduce a transparent and best price-performance scheme for our broad selection of dual labelled probes as follows:

Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol for probes ranging from 15 to 40 nucleotides does not allow for ambiguity in terms of what you expect and pay for.

metabion´s routine dual labeled probe portfolio perfectly covers the spectrum of applicable excitation and detection wavelengths, with reporter-quencher combinations that are suitable for all commonly used real-time PCR platforms. However, do not hesitate to ask for non-listed combinations including internally or multi-labeled oligos and probes. Contact us

Our Prices include HPLC purification as well as Mass-Check QC to ensure you receive the highest-quality product possible.

When you write your email, please make sure to address the following questions in the excel template:

Name of the RNA DLP?

Sequence of the RNA DLP in 5’-3’ orientation?

Yield range

Delivery form? (dry / in water/ buffer + concentration)

Modifications?

If you are a new customer, please additionally provide us with

Your shipping and billing address

Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU) etc

In case you opt to transmit orders via email using your own format(s), we need to alert you that above mentioned information are obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted emails.

Confirmation

Online orders or email orders which indicate an email address will be confirmed by email.

Delivery

Our default shipping mode is sending by Express service overnight at EURO 4.20 per shipment within Germany. If the value of your order is > EURO 125,00 shipping within Germany is free of charge!For other countries please see our shipping table.

Average in-house turnover times (freight forwarders delivery time not included):

double labelled probes: 10-15 working days

Above mentioned estimated turnover times are only indications and refer to our standard portfolio. In terms of "counting" working days, orders placed past 15:00 (Munich time) are considered to be next day's order. Major deviations will be communicated timely. Be assured that we try to process your order as quick as possible without compromising on quality!

The chemistry of RNA synthesis is identical to the DNA synthesis except for the presence of an additional protecting group at the 2' hydroxyl position of ribose. This position is protected with silyl groups (usually TBDMS), which are stable throughout the synthesis. The remaining positions on both the sugar and the bases are protected in the same fashion as in DNA. Please visit our DNA FAQs for more information.

Using our optimized production pipeline, we can deliver RNA DLPs of over 40 bases. The maximum length of an oligonucleotide depends both on the sequence and on the modifications of interest. For example, consider a DLP having a reporter at the 5’-end and a quencher at the 3’-end. The length of the DLP can compromise the activity of the quencher, if the distance between reporter and quencher is too high. Therefore, we consider a length of 40 bases as maximum, for a DLP probe to function optimally.

However, you can now go beyond this limit with our ZNA technology! Want to learn more? Click here.

With this technology, the quencher is brought in closer proximity to the dye and its effect is optimized. You only need to take into account that the Tm of the resulting probe will be higher, compared to that of a native (non-ZNA) probe. To get best performance for your experiments, we can help you designing your probe. Please do not hesitate to contact us!

For RNA oligonucleotide between 5-40 nts and for a quantity delivered between 10-100 nmol, we offer RNA oligonucleotides desalted as well as HPLC purified. Over 100 nmol and 40 nts our default purity is HPLC! Such RNA require HPLC purification to guarantee the top quality required for your experiments. For such RNA oligonucleotides, HPLC purification is included in our price per base/probe/duplex. Visit our Standard Portfolio.

For desalted RNAs falling into the specifications of our standard portfolio, you can expect at least >80% of purity. HPLC-purified RNAs falling within the specifications of our standard portfolio, have typical purity >85%. Please note that most delivered RNAs will have purities >90%.

* Please note that the term “yield range” refers to the final amount of product you will actually receive. Instead, OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:

Synthesis scale refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. This is the same for all manufacturers of synthetic DNA using standard phosphoramidite chemistry. When a synthesis scale of 40 nmole is specified, approximately 40 nmoles of the first base are added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences; hence it is not possible to produce 40 nmoles of full-length product from a 40 nmole scale synthesis. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control. Final yield is the actual amount that we guarantee to deliver.

Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:

Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.

The expected average in-house turnover time is 10-15 working days. Please note that we perform strict quality controls on your ordered RNA DLPs. In case one or more RNA DLPs do not pass our quality control, they will have to be resynthesized. This may, of course, result in a delay.

DNA synthesis is a complicated process, which has improved significantly over the last years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses; however, it is inevitable that we will occasionally have to re-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.

There is a normal degree of variation in the appearance of the supplied dry oligonucleotide pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of DLPs may vary from powdery to hyaloid. The color changes according to the dye attached.

Distilled sterile water pretreated with DEPC, or any nuclease free biological buffer (i.e. with physiological pH) are acceptable as diluents. The recommended diluent volume is 100 µl - 1 ml, the concentration depending on the application to be used and the yield of the resulting product. (see also How stable is my RNA DLP once I have resuspended it?)

The best way to store RNA is as a dry pellet at -20°C to -80°C and to avoid frequent freeze-thaw cycles. If you want to store your RNA in solution re-suspend the delivered pellet in an RNAse-free solution buffered at pH 7.4 - 7.6 and store at -20°C or less. We recommend that RNAs are re-suspended at a convenient stock concentration and stored in small aliquots to avoid multiple freeze thaw cycles.

For modified oligonucleotides – especially for fluorescent dye labelled oligos – you should minimize the probe’s exposure to light because of its bleaching effect. Additionally, we recommend storing dye labelled oligos highly concentrated, rather than in working dilutions, if you don’t use them immediately. The higher the dilution factor the faster the fluorescent activity decreases/fluorescence fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, and dilute them just before usage. Click here to download our guidelines for handling RNA.

Maintaining sterile, RNAse free conditions is always recommended as a precaution. Dried pellets are stable at room temperature for 2-4 weeks, but should be placed at -20°C or -80°C for long term storage. Under these conditions, dry RNA is stable for at least one year.

For correct storing and best performance of your RNA DLP, we recommend the following:

Avoid repeated freeze-thaw, as this will denature the RNA DLP.

Avoid the use of water as a diluent, since its pH may be as low as 4-5.The RNA DLP stability in solution depends on the pH. Dissolving DLPs into acidic solutions may result in oligo degradation. Therefore, avoid the use of non-sterile distilled water as solution pH might be as low as 4-5.

Minimize the exposure of RNA DLPs to light, to avoid any bleaching effect.

Store RNA DLPs highly concentrated and not in working dilutions, if you are not planning to use them within 24 hours. The higher the dilution factor, the faster the fluorescent activity fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, dilute them just before you use the probe and store the aliquots at 4°C in the dark.

For a detail explanation of DLPs and their use in qPCR, please refer to our Learning Platform.

You also need to consider the following:

Sequence Lengthmetabion can routinely synthesize RNA DLPs ranging from 18 to 40 bases. However, consider a DLP having a reporter at the 5’-end and a quencher at the 3’-end. The length of the RNA DLP can compromise the activity of the quencher, if the distance between reporter and quencher is too high. However, you can now go beyond this limit with our ZNA technology! Want to learn more? Click here. For long RNA DLP and special requests, please inquire.

Sequence CompositionMake sure your sequence is free of hairpins and self-complementary regions. Also, more than six of the same consecutive bases (i.e. GGGGGGG) can be problematic and reduce final yields.

In case you need to design probes into difficult, GC-rich regions, you may want to consider replacing dG, with 7-deaza-dG, which destabilizes the formation of secondary structures.

To increase the Tm of your primers/probes you can use base analogues, such as pdC, pdU, 2-Aminopurine.

To improve the efficiency, sensitivity and specificity (via increasing the Tm), you can consider using our ZNA technology. For more information, visit our ZNA page. We can help you designing ZNA primers and probes. Contact us!

Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.

Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as

n-1/n-x oligos

Depurination

Incomplete Deprotection

Acrylonitrile adducts

High Salt Content Identification

Moreover, it is the fastest and most efficient way to identify potential product mix-ups.

We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.

MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).

ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.

Comparison MALDI-ToF and ESI-ToF

Qualification Criteria

MALDI-ToF

ESI-ToF

< 60 nts

+

+

> 60 nts

-

++

Photosensitive Modified Oligos

-

+

Wobble Oligos

-

+

Throughput

++

+

n-1/n-x Detection

+

+

Incomplete Deprotection

+

+

Depurination

+

+

Mass Accuracy

++

++

Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.

For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.

Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.

Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.

metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.

For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?

The label on the RNA DLP tube shows basic information like oligo name, name of person who ordered, DLP sequence including modifications, RNA DLP ID, amount of DNA (OD260 and nmol), Tm, and molecular weight.

In addition, you will receive a technical data sheet containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of DNA (OD260 and nmol), Tm and molecular weight. Additionally, you will receive a hard copy of the Mass Check trace.

The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:

Mass CheckStandard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge.

All oligonucleotides, whether single-stranded or double-stranded oligos, are provided as dried pellets and shipped at ambient temperature. While being stable at room temperature for 2-4 weeks, they should be placed at -20°C or at lower temperature upon receipt.